Endophytic Fungi from Panax Ginseng and Their Secondary Metabolites
|School||Second Military Medical University|
|Keywords||Panax ginseng C. A. Meyer endophytic fungi bioactivity LC/MSanalysis GC/MS analysis secondary metabolites|
Endophytes are microorganisms such as fungi and bacteria (including actinomycete) that live inter- and/or intra-cellularly inside the healthy plant tissues causing asymptomatic infections for all or part of their life cycle. Endophytic fungi have been investigated to be a big reservoir providing a wide variety of structurally unique and/or bioactively strong natural products such as alkaloids, flavonoids, steroids, terpenoids, and so on.In the present study, 48 isolates were isolated from ginseng. By means of their morphological characteristics,phylogenetic analysis and similarity comparison of ITS sequences, 40 isolates were identified into 14 taxa, including 12 species of 10 genera and 2 taxa placed above family level.In this paper, the screening of ginsengnosides from endophytes using HPLC and HPLC-MS is reported. The evidence from SIM and SCAN screening demonstrated that there’s no ginsengnosides could be detected from endophytes.Then, based on the mircodilution method, antifungal active endophytes were fast screened by Pyricularia oryzae P-2b model, and activities of endophytes against pathogenic fungi and tumor cells were tested. Thus, 33.3 % of endophytic fungi fully suppressed the activity of P. oryzae P-2b. And, 11 colonies (22.9 %) showed excellent antifungal activities against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum and Aspergillus fumigatus while 5 colonies (10.4 %) showed excellent antitumor activities against MKN45, LOVO, HepG2 and HL-60 tumor cells. Among the bioactive colonies, the grows of Trichophyton rubrum was strongly inhibited by Yuan-25(Penicillium melinii), as the MIC80 was equal to the positive control fluconazole, while the IC50 of Yuan-27(Mortierella monospora) were all similar with Doxorubicin.Among the strains isolated, Yuan-25(Penicillium melinii), a stable colony with special morph aroused our interests, moreover it excreted oil-like yellow liquids and emitted special smells, indicate that it may be abundant in ether extract components. By means of GC/MS technique, some bioactivitive compounds were determined, such as xanthatin, isotanshinoneⅡetc. It is attractive that the extracts derived from the endophyte and ginseng samples contained the same compound falcarinol, a natural pesticide and anti-cancer agent.A total of 14 compounds were isolated from strains as Yuan-13(Penicillium sp.), Yuan-25(Penicillium melinii), Yuan-27(Mortierella monospora), Yuan-30 and YSS-13 (Alternaria mali) by the column chromatography on Sephadex LH-20 and so on. The structures of 10 compounds were mainly elucidated by using spectroscopic methods (1H-NMR, 13C-NMR, DEPT, HSQC, HMBC and ESI-MS etc.). One novel with 8 known compounds were isolated from the active fractions. These structures were identified to be (1) 2,4-Dihydroxy-3-methyl-6-[(Z)-(6-methyl-5,6-dihydro-2H-pyran-2- ylidene)methyl]-benzaldehyde; (2) Brefeldin A; (3) 2,4,5-trimethyl-1,3-benzenediol; (4) 2,4-dihydroxy-3,5,6-trimethyl-methyl benzoate; (5)mannitol; (6) Penicillic acid; (7) ergosterol; (8) ergosterol peroxide; (9)2-(4-Hydroxyphenyl)ethanol ( tyrosol). Bioassay results further indicated that 2,4-Dihydroxy-3-methyl-6-[(Z)-(6- methyl-5,6-dihydro-2H-pyran-2-ylidene)methyl]-benzaldehyde and Brefeldin A have excellent antitumor properties.In summary, we studied the isolation, identification and bioassay of endophytes, the extraction and separation of their components, and the comparative study of the bioactive strain Yuan-25(Penicillium melinii) and ginseng, etc. Endophytes are a potential source of novel anti-microbe chemistry and biology to assist in helping solve not only human health, but plant and animal health problems also. Indeed, it showed that endophytic fungus was isolated from ginseng root have a wide range of antifungal and antitumor activities. Our study is part of a wider project aimed at the possible exploitation of the endophytic fungi isolated from ginseng. Research on the endophytes not only brings vast social profits but also has realistic theoretic significance.